THE DNAB PROTEIN OF ESCHERICHIA-COLI - MECHANISM OF NUCLEOTIDE BINDING, HYDROLYSIS, AND MODULATION BY DNAC PROTEIN
THE DNAB PROTEIN OF ESCHERICHIA-COLI - MECHANISM OF NUCLEOTIDE BINDING, HYDROLYSIS, AND MODULATION BY DNAC PROTEIN
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DOI：
10.1021/bi00371a019
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发表时间：
1986-11-18
期刊：
影响因子：
2.9
通讯作者：
BISHOP, JE
中科院分区：
文献类型：
--
作者：
BISWAS, EE;BISWAS, SB;BISHOP, JE
利用荧光核苷酸类似物研究了dnaB蛋白和dnaB.cntdot. dnac蛋白复合物结合和水解核苷酸的机制。三磷酸三硝基苯基腺苷（TNP-ATP）或相应的二磷酸（TNP-ADP）的结合会导致发射最大值的蓝移和核苷酸类似物荧光发射的数倍放大。对TNP-ATP结合的Scatchard分析表明，TNP-ATP结合具有高亲和力（Kd = 0.87 .mu）。M)，核苷酸的荧光发射增强8.5倍。只有三个TNP-ATP或TNP-ADP分子与dnaB蛋白的六聚体相结合，而ATP或ADP分子与dnaB六聚体相结合。TNP-ATP和TNP-ADP都是单链（SS） dna依赖性atp酶活性的竞争性抑制剂。TNP-AMP既不与dna蛋白结合，也不抑制atp酶活性。dnaC蛋白形成dnaB.cntdot.dnaC复合物，导致TNP-ATP荧光增强减弱，同时SS dna依赖性atp酶活性降低。dnaB.cntdot.dnaC复合物atp酶活性的动力学分析表明，复合物形成过程中atp酶活性的降低是由于最大速度（Vmax）的降低。然而，当单链M13 DNA存在时，dnaB蛋白水解TNP-ATP和dATP的速率极慢。核苷酸的2”-OH基团很可能在水解反应中起重要作用，但在核苷酸结合中不起作用。
The mechanism of nucleotide binding and hydrolysis by dnaB protein and dnaB.cntdot.dnaC protein complex has been studied by using fluorescent nucleotide analogues. Binding of trinitrophenyladenosine triphosphate (TNP-ATP) or the corresponding diphosphate (TNP-ADP) results in a blue shift of the emission maximum and a severalfold amplification of the fluorescence emission of the nucleotide analogues. Scatchard analysis of TNP-ATP binding indicates that TNP-ATP binds with a high affinity (Kd = 0.87 .mu.M) and a 8.5-fold enhancement of fluorescence emission of the nucleotide. Only three molecules of TNP-ATP or TNP-ADP bind per hexamer of dnaB protein in contrast to six molecules of ATP or ADP binding to a dnaB hexamer. TNP-ATP and TNP-ADP are both competitive inhibitors of single-stranded (SS) DNA-dependent ATPase activity of dnaB protein. TNP-AMP neither binds to dnaB protein nor inhibits the ATPase activity. Formation of dnaB.cntdot.dnaC complex by dnaC protein results in diminution of the TNP-ATP fluorescence enhancement and a concomitant decrease in the SS DNA-dependent ATPase activity. Kinetic analysis of the ATPase activity of dnaB.cntdot.dnaC complex indicates that the decrease in the ATPase activity on complex formation is due to a reduction of the maximal velocity (Vmax). The dnaB protein hydrolyzes both TNP-ATP and dATP, however, with an extremely slow rate in the presence of single-stranded M13 DNA. The 2''-OH group of the nucleotide most likely plays an important role in the hydrolysis reaction but not in the nucleotide binding.